Genetic basis of praziquantel resistance Mass treatment campaigns using praziquantel (PZQ) monotherapy impose strong selection for drug resistance in schistosome parasites. Resistance can be selected in the laboratory, and resistant parasites have been isolated from patients, but the current level of resistance in natural schistosome populations is not possible to quantify using classical parasitological methods. This proposal aims to identify the gene(s) underlying PZQ resistance in order to (a) understand the mode of action of this drug, (b) determine the mechanism of resistance, and (c) develop molecular markers for field surveillance of resistance in treatment programs. In preliminary work (funded by R21 AI096277), we conducted genetic crosses between sensitive and resistant parasites (generated by PZQ selection in the laboratory) and used exome sequencing to characterize F2 parasites that survived or died following PZQ treatment. We identified multiple markers linked to PZQ resistance on 4 scaffolds spanning 4.29Mb and containing 110 genes. These scaffolds are assigned to chr 3 but are currently unassembled in the genome sequence. In Aim 1, we will fine map the gene(s) involved in PZQ resistance (i) by correctly positioning the unassembled scaffolds to the genome sequence by exome sequencing and linkage analysis of SNP segregation in an existing genetic cross, (ii) by systematic RNAi knockdown of prioritized genes in the QTL region and phenotypic analysis of adult parasites, and (iii) by transfection-based manipulation of candidate genes. In Aim 2, we will examine the relevance of this genome region to PZQ resistance in the field. To do this we will compare genome-wide allele frequencies in larval parasite (miracidia) populations collected from schistosome-infected patients before and after PZQ treatment from sites in Kenya and Uganda. These analyses will identify genome regions showing systematic change in allele frequencies in parasites surviving PZQ treatment. This information will be invaluable for control programs. As a complementary approach, in Aim 3 we will sequence exomes of individual worms from an archived collection of frozen PZQ-resistant parasites previously isolated from the field and selected in the laboratory. These data will critically examine the hypothesis that genes in the chr. 3 scaffolds are enriched in such parasites when compared to sensitive parasites of population controls. We have previously demonstrated the power of combining genetic linkage mapping studies, functional analyses and analysis of field-collected parasites for determining the genetic basis of oxamniquine resistance. Parallel approaches promise to be equally effective for determining the genetic basis of PZQ resistance.